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J. Vet. Sci.
(2004),
/
5
(2), 173–180
Canine biphasic synovial sarcoma: case report and immunohistochemical
characterization
Panayiotis Loukopoulos
2,#,
*, Hock Gan Heng
1,3
, Habibah Arshad
4
Division of Veterinary Pathology and Anatomy, School of Veterinary Science, The University of Queensland,
St. Lucia 4072, Australia
1
Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia
The clinical, radiological and pathologic features of a
biphasic synovial sarcoma in the left elbow joint of a two-
year-old male Rottweiler are presented. The tumor
showed positive immunoreactivity for vimentin, Epithelial
Membrane Antigen (EMA), p53 and PCNA, while it was
negative for the cytokeratin used, S-100, Rb and p21.
Immunohistochemistry for EMA allowed the identification
of epithelioid components of synovial sarcoma, and may,
therefore, contribute in establishing a diagnosis of
biphasic synovial sarcoma. Intratumoral variation in
PCNA immunoreactivity was minimal, indicating that the

various tumor components proliferate at more or less similar
rates. Overall, the characterized immunohistochemical
profile for canine synovial sarcoma, not defined
previously, may provide clues to the histogenesis of the
phenotypically mesenchymal and epithelial elements of
the tumor, and may be of value in the differential
diagnosis of challenging cases, decreasing the risk of
under- and mis-diagnosis. Although more cases need to be
studied to determine whether there is a consistent pattern
of immunostaining in canine synovial sarcoma, its
potential significance is discussed in relation to the
histogenesis, molecular pathology and differential
diagnosis of canine synovial sarcoma.
Keywords:
Epithelial Membrane Antigen, dogs, immunohis-
tochemistry, neoplasms, p53, radiology, pathology, biphasic
synovial sarcoma
Introduction
Tumors affecting joints are rare, and most are malignant
rather than benign. Those reported in the veterinary literature
are almost exclusively synovial sarcomas affecting dogs
[2,37]. Synovial sarcoma has been reported infrequently in
cats, cattle, horses and other species [33], although it accounts
for about 8% of all soft-tissue sarcomas in humans [33]. In
dogs, it most frequently involves the stifle and elbow, although
other sites, including, for example, a rare case with bilateral
hip joint involvement, have been reported [17].
Despite the assumed mesenchymal origin of the tumor
and a morphological similarity with normal synovial tissue
lining joints and tendon sheaths, the histogenesis of synovial

sarcoma is not clearly defined [10,18,23]. Microscopically,
it may be characterized by a monophasic or biphasic cellular
pattern; the biphasic pattern is diagnostically more distinct
and comprises of a sarcomatous component and an
epithelioid component, which may form clefts and
pseudoacini [25]. Synovial sarcoma may therefore resemble
malignant fibrous histiocytoma, fibrosarcoma, giant-cell
tumor of soft tissue or other tumors [5,31]. Consequently, it
may present a diagnostic challenge for some pathologists
and may be underdiagnosed, particularly atypical or
monophasic variants, without the classic biphasic features,
or cases with metaplastic bone formation or calcification
zones. It is particularly important to differentiate synovial
sarcomas from osteosarcomas, a defining feature of which is
osteoid production by the malignant cells, because the latter
tend to metastasize to the lungs and lymph nodes later in the
clinical course of the disease [25,32].
Similarly, while the possible role of certain oncogenes and
tumor suppressor genes [5], such as
β
-catenin [14] and p53
[28], in the pathogenesis of human synovial sarcoma has
been reported, the molecular pathology of canine synovial
sarcoma has not studied extensively or understood.
A specific immunohistochemical profile for canine
synovial sarcoma has not been clearly defined previously,
and reports on various epitopes are sparse [1]. The
*Corresponding author
Phone: +81-3-3542-2511 ext 4210; Fax: +81-3-3248-2463
E-mail: and

#
Current address
2
Pathology Division, National Cancer Center Research Institute, 5-1-1
Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
Phone: +81-3-3248-2463
E-mail: and
3
Department of Radiological Health Sciences, Colorado State University,
Fort Collins, CO 80523, USA
4
Department of Veterinary Clinical Sciences, Royal Veterinary College,
Hawkshead Lane, North Mymms, Hatfield AL9 7TA, United Kingdom
174 Panayiotis Loukopoulos
et al.
development of such a profile may provide clues to the
histogenesis of the phenotypically mesenchymal and
epithelial elements of the tumor, while, at the same time, it
may be of value in the differential diagnosis of challenging
cases, decreasing the risk of under- and mis-diagnosis.
Immunohistochemical detection of Epithelial Membrane
Antigen (EMA), for example, may allow the identification
of epithelioid components of human synovial sarcoma [27],
although immunoreactivity to EMA has not been reported in
canine tissues.
In the case presented here, the clinical, radiological and
pathologic features of a biphasic synovial sarcoma in a
young dog are described, the tumor is characterized using
immunohistochemistry and histochemistry, and the
immunohistochemical profile of the tumor is discussed in

relation to the histogenesis, pathogenesis and differential
diagnosis of canine synovial sarcoma.
Materials and Methods
A two-year-old male Rottweiler was referred to the
University Veterinary Hospital, Universiti Putra Malaysia
for evaluation of progressive lameness of the left forelimb of
4 months duration. Clinical, cytological and radiological
examinations, and following the animal's euthanasia, the
post mortem and histopathological examination were
performed in a routine fashion.
Immunohistochemistry was performed on formalin-fixed,
paraffin-embedded, silane-coated slides employing a
streptavidin-biotin-peroxidase protocol as described
previously [15,21], counterstained with haematoxylin. The
antibodies used were: vimentin (V9 antibody, 1 : 400
dilution), cytokeratin (MNF116, 1 : 800), S-100 (1 : 400),
Proliferating Cell Nuclear Antigen (PCNA, PC-10, 1 : 800),
Epithelial Membrane Antigen (EMA, E29) (all DAKO,
Carpinteria, USA), p21 protein (NCL-WAF1, 1 : 30),
Retinoblastoma susceptibility gene protein (NCL-Rb, 1 : 50,
both Novocastra), and p53 protein (CM-1 antibody, 1 : 75,
Signet Laboratories, USA). The slides were subjected to ten
minutes of microwave heating (low setting) in a citrate
buffer, pH 6. Positive controls were neoplastic or normal
tissues known to contain the relevant epitope [7,36]. The
primary antibody was substituted with non-immune sera or
Tris buffer in the negative controls.
Results
History and clinical examination
A two-year-old male Rottweiler was referred to the

University Veterinary Hospital, Universiti Putra Malaysia
for evaluation of progressive lameness of the left forelimb of
4 months duration. It had no history of trauma and many
veterinarians had treated it with different types of non-
steroidal anti-inflammatory drugs, without improvement of
the lameness. Upon physical examination, the dog was
depressed, and having non-weight bearing lameness of the
left fore limb. Severe muscle atrophy of the limb was
observed. Pain was evident upon palpation and manipulation
of the elbow joint. There was also evidence of soft tissue
swelling around the joint. Neurological examination
revealed no abnormalities.
Initial radiological examination
The mediolateral radiograph of the left elbow revealed
generalized reduced density of the distal humerus and also
the proximal radius and ulna. The trabecular pattern of the
olecranon was ill defined. Cortical destruction of the cranial
border of the medial epicondyle as well as the cranial border
of the lateral epicondyle was observed. Cortical destruction
of the proximal cranial border of the radius was also present
(Fig. 1). The cranio-caudal view of the elbow showed that
there was a focal rounded lytic area, approximately 2 cm in
diameter, at the supratrochlear foramen of the humerus. Two
foci of cortical destruction, of the lateral and medial
epicondyles of the humerus respectively, were also observed
(Fig. 2). There was evidence of soft tissue swelling in both
radiographs. Thoracic radiography was unremarkable.
Management
Following this, fine needle aspiration was carried out, but,
unfortunately, only numerous erythrocytes and few leucocytes

were observed on cytological examination. No conclusive
diagnosis was made. The tentative diagnoses at this point
included synovial sarcoma, deep fungal infection and
metastatic neoplasia. Recommendations to the owner
included core biopsy of the lesion, amputation of the limb or
F
ig. 1. Mediolateral radiographic view of the left elbo
w.
G
eneralized reduced density of the distal humerus and also t
he
p
roximal radius and ulna. The trabecular pattern of the olecran
on
i
s ill defined. Note the cortical destruction of the cranial border
of
m
edial epicondyle, the cranial border of lateral epicondyle, a
nd
t
he cortical destruction of the proximal cranial border of t
he
r
adius.
Canine biphasic synovial sarcoma: case report and immunohistochemical characterization 175
euthanasia. In view of the guarded prognosis, the owner
decided to bring the dog back home for a few days before
euthanasia. Enrofloxacin (Baytril, Bayer, Germany) 5 mg/kg
once a day and ketoprofen (Ketofen, Merial, France) 1 mg/kg

once a day for 5 days were dispensed, to prevent secondary
bacterial infection following the fine needle aspiration and to
alleviate pain.
Clinical and radiological re-evaluation
After two weeks, the dog was presented again. The owner
reported that there was no improvement of the dog’s
condition. Left elbow radiography was carried out again to
determine the progression of the disease. The mediolateral
view of the left elbow revealed that there was further cortical
destruction of the cranial border of the medial and lateral
epicondyle and the proximal cranial border of the radius
(Fig. 3). The craniocaudal view radiograph showed that the
focal lytic area had expended and that it had a diameter of
about 2.5 cm (Fig. 4). The lateral epicondyle was nearly
destroyed. Euthanasia was performed on the request of the
owner. The reason given was that the owner could not bear
having a three-legged dog and the agony over the possibility
of the development of tumor metastases.
Gross pathology
The post-mortem examination was carried out
immediately after euthanasia. Grossly the lesion extended
from the distal humerus to the proximal radius and ulna. An
irregular lobulated white mass, of approximately 2.5 cm in
diameter, was found at the medial condyle of the humerus
(Fig. 5). Another mass, hemorrhagic in appearance, was
found in the elbow joint (Fig. 6). The cancellous bones of
the distal humerus and olecranon were destroyed. The lungs
were normal upon examination. There were no significant
findings in other organ systems. Multiple tissue samples
from different parts of the lesions and various organs were

taken for histopathological examination.
Histopathology
Histopathologic examination was based on multiple tissue
sections covering different parts of the tumor. The tumor
appeared well circumscribed, unencapsulated and in
intimate relationship to synovial structures. The mass was
composed of highly pleomorphic cells, arranged in various
morphological patterns. These mainly included cellular
areas consisting of sheets of roundish to polygonal cells and
areas consisting mostly of spindle cells arranged in a
storiform pattern (Figs. 7 and 8).
In the former area, the dense stroma of cells did not
F
ig. 2.
Craniocaudal radiographic view of the left elbow. A foc
al
r
ounded lytic area at the supratrochlear foramen of the humeru
s,
a
nd two foci of cortical destruction of both the medial and later
al
e
picondyles are shown.
F
ig. 3.
Mediolateral radiographic view of the left elbow. Day 1
4:
f
urther cortical destruction of the cranial border of the medial a

nd
l
ateral epicondyle and the proximal cranial border of the radius
.
F
ig. 4.
Craniocaudal radiographic view of the left elbow. Day 1
4:
T
he focal lytic area has expended; the lateral epicondyle is near
ly
d
estructed.
176 Panayiotis Loukopoulos
et al.
produce any considerable amount of extracellular matrix,
apart from small quantities of collagen. Small to medium-
sized scattered necrotic foci covered approximately 30% of
the area and were associated with hemorrhage. Cells were
large, round, polygonal or, less frequently, spindle-shaped
with distinct mauve cytoplasm and eccentric nuclei. The
nuclear:cytoplasmic ratio was moderate. Cellular features
included karyomegaly, anisokaryosis and bizarre mitoses;
nuclei were round to oval in most cells or elongated in the
spindle cells. Epithelioid cells with vesicular nuclei were
occasionally encountered. A large proportion of cells had
one or two prominent nucleoli and stippled chromatin
pattern. There was a considerable number of bi- and tri-
nucleated cells and giant mononucleated cells; the nuclei of
the multi- and mono-nucleated cells were similar. Apoptosis

was moderate while the mitotic index varied from 2 to 7 per
high power field (average 3.3).
The latter areas of the tumor were reminiscent of
malignant fibrous histiocytoma, characterized by whorling
and streaming patterns of indistinctly bordered elongated
cells and rather dense collagen fibers. Anisokaryosis was
marked, mitotic index was lower in this area (2.5/high power
field), while there were no necrotic foci.
In other areas, a mixed population of round or small
fibroblast-like cells formed streams or packets of cells in a
loose mucin-like matrix or collagen. A small number of slits
and clefts lined by malignant cells were noticed. Mitoses,
multinucleated and giant cells were few.
No osteoid or chondroid production were observed. There
was evidence of osseous invasion by the tumor cells,
including the medullary cavity. The lymph nodes examined
F
ig. 5.
Gross pathology: a lobulated mass at the medial condy
le
o
f the humerus.
F
ig. 6.
Gross pathology: hemorrhagic mass found in the elbow joi
nt.
F
ig. 7.
Synovial sarcoma. Mixed tumor cell population, includi
ng

s
pindle, epithelioid and multinucleated cells (H & E
×
200).
F
ig. 8.
Synovial sarcoma. Pleomorphic tumor cell populatio
n;
c
ells with one or two prominent nucleoli, abnormal mito
tic
f
igures and apoptotic bodies are shown (H & E
×
400).
Canine biphasic synovial sarcoma: case report and immunohistochemical characterization 177
did not appear to be affected. The above elements allowed
the diagnosis of biphasic synovial sarcoma to be established.
Overall, based on the degree of nuclear pleomorphism
(marked), the number of mitoses per 10 high power (400x)
fields [21] and the proportion of tumor necrosis (<10%
overall), the tumor was classified as Grade II[35].
Histochemistry and immunohistochemistry
Tumor sections stained with Alcian Blue allowed the
detection of mucin in the interstitial matrix, while staining
following a PAS reaction was unremarkable.
More than 80% of the tumor cells showed intense
cytoplasmic staining for vimentin, while the majority of
them also showed strong nuclear staining for PCNA (Fig. 9).
p53 was detected immunohistochemically in 10-20% of

tumor cells that showed moderate to intense nuclear staining
(Fig. 9). Foci of cells positive to EMA were observed (Fig.
10). Tumor cells were uniformly negative to the cytokeratin
used, as well as to the Rb, p21 and S-100 antibodies (Fig.
11).
Discussion
Synovial sarcoma is a rare tumor in dogs [37], that may
present histologically as a monophasic (mesenchymal) or a
biphasic (mesenchymal and epithelial) variant. The
histogenesis, pathogenesis and immunohistochemical
profile of canine synovial sarcoma have not been clearly
defined [10,18,23], and the veterinary literature on the
subject is sparse [1]. In the case presented here, the clinical,
radiological and pathological features of a biphasic synovial
sarcoma in a young dog are described. The tumor is
characterized using immunohistochemistry and
histochemistry, and the derived immunohistochemical
profile is discussed in relation to the histogenesis and
differential diagnosis of canine synovial sarcoma.
Synovial sarcoma occurs mainly in male, middle-aged
large breed dogs [22]; our patient, however, was only two
years old. Progressive lameness is a clinical sign common to
all cases. The duration of the lameness generally ranges
from several weeks to months, although longer periods have
been recorded [22]. In this case, the dog was progressively
lame for 4 months and, upon presentation, it was not weight
bearing, due to pain in the elbow joint.
Presence of a periarticular soft tissue mass may be the
only radiographic finding and an ill-defined periosteal
reaction with some cortical thinning may be the earliest

detectable skeletal change [20]. This progresses to more
obvious areas of trabecular and cortical lysis with poorly
defined margins [11]. The tumor commonly crosses the
joint, resulting in involvement of adjacent bones [22].
Beside the presence of periarticular soft tissue mass, the
interesting radiographic features of this case include the
F
ig. 11.
Immunohistochemical localization of Epithel
ial
M
embrane Antigen: diffuse, moderately intense cytoplasm
ic
s
taining of tumor cells. Streptavidin-biotin-peroxidase, Maye
r’s
h
ematoxylin counterstain.
×
400.
F
ig. 9.
Immunohistochemical localization of PCNA: inten
se
n
uclear staining of the majority of tumor cells. Streptavidi
n-
b
iotin-peroxidase, Mayer’s hematoxylin counterstain.
×

400.
F
ig. 10.
Immunohistochemical localization of p53 protei
n:
m
oderately intense nuclear staining of tumor cells. Streptavidi
n-
b
iotin-peroxidase, Mayer’s hematoxylin counterstain.
×
200.
178 Panayiotis Loukopoulos
et al.
aggressiveness of the osteolytic lesion of the humerus and
the cortical thinning of the epicondyles. The periosteal
reaction was minimal.
Most patients with synovial sarcomas develop metastatic
tumors [35]. Amputation, localized resection of the tumor
and chemotherapy are the options of treatment for synovial
sarcoma [35]. Amputation of the involved limb yields a
better prognosis as most dogs have disease-free interval and
survival time of more than 36 months. Local resection is
invariably followed by recurrence at the site and then
amputation is the appropriate recourse [35]. The outcome of
chemotherapy in the treatment of synovial sarcoma in dogs
is not good; although various chemotherapeutics have been
reported to have efficacy as adjuvants in treating synovial
sarcoma in human beings, there is only one report of a
positive response to treatment in the dog, with a

combination of cyclophosphamide and doxorubicin [34]. In
the case presented here, however, the owner opted for
euthanasia, despite the good prognosis for amputation, and
the absence of radiographic evidence of lung metastasis of
the tumor upon presentation.
On histopathological examination, osteoid production by
the malignant cells, a defining feature of osteosarcoma, a
tumor entity encountered much more frequently in practice,
was not observed in our case. On examination of one tumor
area alone, however, the dominant presence of a storiform
pattern or multinucleated cells meant that the diagnosis of
malignant fibrous histocytoma or giant cell tumor of bone
could be entertained. When multiple sections were
examined, however, the typical biphasic pattern of the
tumor, combined with the clinical, radiological, and
immunohistochemical findings, rendered the diagnosis of
synovial sarcoma. The histochemical stain for Alcian Blue
helped confirm the diagnosis.
The immunohistochemical profile of the tumor was
determined, and was compatible with and suggestive of a
diagnosis of synovial sarcoma. The positivity of the tumor
cells for vimentin was indicative of their mesenchymal
derivation [29]. Tumor cells were uniformly negative to the
cytokeratin used. The above are in agreement with a recent
study, in which all synovial sarcomas stained positive for
vimentin and negative for cytokeratins [8]. Despite being
negative to cytokeratin, foci of cells positive to Epithelial
Membrane Antigen (EMA) were observed. EMA
immunohistochemistry may allow the identification of
epithelioid components of synovial sarcoma [27], and in our

case, tends to confirm the biphasic nature of the tumor,
although a diagnosis of osteosarcoma should not be ruled
out on the basis of EMA immunoreactivity alone [16]. To
our knowledge, this is the first time that positive
immunoreactivity to EMA is reported in canine tissues.
S-100 is a protein that serves as a marker for bone tumors
originating in the cartilage, the notochord and T-zone
histiocytes and is also involved in the calcification of normal
and neoplastic cartilage [4,24]. The fact that the present case
was S-100 negative further supports our previous
observations concerning the histogenesis of the tumor.
Proliferating Cell Nuclear Antigen (PCNA) acts as an
auxiliary protein for DNA-polymerase delta and is increased
in proliferating cells as opposed to mitotically quiescent
cells [38]. It serves as a proliferation marker [6] and has
been shown to be of prognostic value for a number of tumor
types [26]. The majority of tumor cells in our case showed
strong nuclear staining for PCNA. Interestingly,
intratumoral variation in PCNA immunoreactivity was
minimal, indicating that the various tumor components
proliferate at more or less similar rates.
p53 was detected immmunohistochemically in 10-20% of
tumor cells that showed moderate to intense nuclear
staining. The p53 gene normally acts as a tumor suppressor
gene [9]. p53 guards cells against replication when their
genome is abnormal, by arresting the cell cycle and by either
activating and regulating DNA repair, or by inducing
programmed cell death (apoptosis) if genomic damage is
excessive [9]. Because of its central role in the cell cycle and
in carcinogenesis [9], p53 is the most frequently altered gene

in human tumors [13]. Immmunohistochemical detection of
p53 protein, as in the present case, demonstrates alterations
in the p53 gene or product [19] which, therefore, appears to
be involved in the pathogenesis of canine synovial sarcoma.
However, the exact mechanisms of p53 involvement and
progression toward a tumor-associated phenotype in
synovial sarcoma would require further molecular studies in
order to be elucidated.
Mutational inactivation of the retinoblastoma susceptibility
gene (Rb) has been proposed as a crucial step in the
formation of retinoblastoma and other tumors, including
human synovial sarcoma and osteosarcoma [3,30]. p21
protein, the product of WAF1/CIP1 gene, is an inhibitor of
cyclin-dependent kinases and a critical downstream effector
in the p53 pathway. The expression of p21 in human
neoplasms may be related to p53 functional status [12].
In our case, neither Rb nor p21 were expressed
immunohistochemically, however, a role for them in the
pathogenesis of synovial sarcoma cannot be ruled out.
The immunohistochemical profile of a case of canine
synovial sarcoma was defined and its potential significance
in relation to the molecular pathology and histogenesis of
canine synovial sarcoma discussed. Overall, the case
presented here was positive for vimentin, EMA, p53 and
PCNA, while it was negative for the cytokeratin used, S-100,
Rb and p21. Immunohistochemistry for Epithelial
Membrane Antigen, in particular, may confirm the biphasic
nature of the tumor, by allowing the identification of
epithelioid components of synovial sarcoma, and may,
therefore, contribute in establishing a diagnosis of biphasic

synovial sarcoma. More cases will need to be studied by this
technique, however, in order to determine whether there is a
Canine biphasic synovial sarcoma: case report and immunohistochemical characterization 179
consistent pattern of staining in synovial sarcomas of the
dog and whether this pattern will be adequately specific to
be of value in the differential diagnosis of synovial sarcoma.
Acknowledgments
We are grateful to Dr.S.Jasni, Faculty of Veterinary
Medicine, Universiti Putra Malaysia; Professor
W.F.Robinson and Dr.S.Yeomans, School of Veterinary
Science, University of Queensland for constructive
comments on histopathology, and Mr.M.Walsh, Department
of Pathology, School of Medicine, University of Queensland
for technical advice on EMA immunohistochemistry.
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